LOCUS Exported 11117 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION All-in-one CRISPR plasmid for integrating, deleting, or replacing a gene in S. cerevisiae. High-copy, URA3 plasmid contains human codon-optimized Cas9 and a guide RNA targeting ARS805a sequence TTATTTGAATGATATTTAGT in yeast chromosome 8.. ACCESSION . VERSION . KEYWORDS p426_Cas9_gRNA-ARS805a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11117) AUTHORS Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A TITLE A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. JOURNAL Nucleic Acids Res. 2017 Jan 9;45(1):496-508. doi: 10.1093/nar/gkw1023. Epub 2016 Nov 28. PUBMED 27899650 REFERENCE 2 (bases 1 to 11117) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11117 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 69..1411 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 401..448 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1438..1542 /gene="bla" /label=AmpR promoter CDS 1543..2403 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1761..1780) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2574..3162 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3063..3082 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3316..3333 /label=L4440 /note="L4440 vector, forward primer" promoter 3486..3516 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3524..3540 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 3529..3551 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3548..3564 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3548..3564 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3583..3603 /label=T3 /note="T3 promoter, forward primer" promoter 3585..3603 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_RNA 3830..3905 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 3991..4695 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 4705..8808 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 8821..8841 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 8857..9104 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(9122..9141) /label=T7 /note="T7 promoter, forward primer" promoter complement(9123..9141) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9151..9168) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9151..9167) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9160..9182) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 9312..9767 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9399..9418) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9609..9630 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(9898..10701) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" promoter complement(10702..10917) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(10948..10967) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 11067..11089 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3601 agggaacaaa agctggagct agtatactct ttcttcaaca attaaatact ctcggtagcc 3661 aagttggttt aaggcgcaag actgtaattt atcactacga aatcttgaga tcgggcgttc 3721 gactcgcccc cgggagagat ggccggcatg gtcccagcct cctcgctggc gccggctggg 3781 caacaccttc gggtggcgaa tgggactttt tatttgaatg atatttagtg ttttagagct 3841 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 3901 ggtgcttttt ttattttttg tcactattgt tatgtaaaat gccacctctg acagtatgga 3961 acgcaaactt ctgtctagtg gatagtcgac atccttttgt tgtttccggg tgtacaatat 4021 ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat accttcgttg 4081 gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata ccagacaaga 4141 cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg gtacataacg 4201 aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc actacccttt 4261 ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc tttttttttc 4321 ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa tgatggaaga 4381 cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg 4441 atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct 4501 ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaaagt 4561 ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg tttcctcgtc 4621 attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca agctatacca 4681 agcatacaat caacttggaa aaaaatggac aagaagtact ccattgggct cgatatcggc 4741 acaaacagcg tcggttgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc 4801 aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg 4861 ttcgactccg gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat 4921 acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag 4981 gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag 5041 cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac 5101 ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg 5161 ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg 5221 gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac 5281 aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg 5341 agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag 5401 aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt 5461 aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat 5521 gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg 5581 gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc 5641 accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg 5701 actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc 5761 gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt 5821 tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag 5881 cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac 5941 cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt 6001 ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta 6061 ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc 6121 atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc 6181 gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct 6241 ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa 6301 gggatgagaa agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc 6361 ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt 6421 gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga 6481 acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac 6541 gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt 6601 gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag 6661 aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac 6721 aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac 6781 ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa 6841 gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct 6901 atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga 6961 aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag 7021 ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg 7081 tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac 7141 ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg 7201 ctctccgact acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt 7261 gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca 7321 gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc 7381 acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat 7441 aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc 7501 caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag 7561 gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt 7621 tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg 7681 gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac 7741 tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc 7801 accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg 7861 gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc 7921 gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg 7981 aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg 8041 aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc 8101 ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg 8161 aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca 8221 agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa 8281 gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga 8341 atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac 8401 gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat 8461 gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa 8521 ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct 8581 gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg 8641 tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac 8701 agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt 8761 acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac 8821 cccaagaaga agaggaaggt gtgatctctt ctcgagtcat gtaattagtt atgtcacgct 8881 tacgttcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 8941 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 9001 ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 9061 ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg tacccaattc 9121 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg 9181 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 9241 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 9301 cgaatggcgc gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 9361 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 9421 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 9481 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 9541 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 9601 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 9661 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 9721 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcct gatgcggtat 9781 tttctcctta cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta 9841 aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta 9901 gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc 9961 ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga 10021 tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc 10081 atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat 10141 gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt 10201 acccttagta tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa 10261 aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc 10321 tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc 10381 cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag 10441 taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa 10501 atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac 10561 taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt 10621 ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc 10681 cttatatgta gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag 10741 ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac 10801 caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa 10861 aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaattg 10921 aaaagctgtg gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 10981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 11041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 11101 atcaccgaaa cgcgcga //